THE TONOPLAST H-ATPASE OF ACER-PSEUDOPLATANUS IS A VACUOLAR-TYPE ATPASE THAT OPERATES WITH A PHOSPHOENZYME INTERMEDIATE()

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure in cluded size-exclusion and ion-exchange chromatography. The H+-ATPase c onsists of at least eight subunits, of 95, 66, 56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis thaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit o f the vacuolar H+-ATPase of oat roots. The functional molecular size o f the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+- ATPase was rapidly phosphorylated by [gamma-P-32]ATP in vitro. The com plete loss of radioactivity in the 66-kD subunit after a short pulse-c hase experiment with unlabeled ATP reflected a rapid turnover, which c haracterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to h ydroxylamine and alkaline pH. These results lead us to suggest that th e tonoplast H+-ATPase of A. pseodoplatanus is a vacuolar-type ATPase t hat could operate with a plasmalemma-type ATPase catalytic mechanism.